An adequate supply of vitamin D is vital as the term “vitamin” already suggests. A deficiency of vitamin D leads to severe diseases such as rickets or osteoporosis. While vitamin D was still regarded as a single substance at the beginning of the last century, the vitamin D system has changed in the course of the last three decades into a complex and manifold network of vitamin D metabolites. Nowadays more than 40 different vitamin D metabolic products are known (Zerwekh, J. E., Ann. Clin. Biochem. 41 (2004) 272-281).
Humans can only produce D3 vitamins or calciferols by the action of ultraviolet rays from sunlight on the skin. Vitamin D3 that is produced in the skin binds to the so-called vitamin D-binding protein which transports it into the liver where it is converted into 25-hydroxyvitamin D3 by 25-hydroxylation. A multitude of other tissues are nowadays known to be involved in vitamin D metabolism in addition to the skin and liver, the two organs that have already been mentioned (refer to Schmidt-Gayk, H. et al. (eds.), “Calcium regulating hormones, vitamin D metabolites and cyclic AMP”, Springer Verlag, Heidelberg (1990) pp. 24-47). 25-Hydroxyvitamin D and more specifically 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 are the central storage form of vitamin D in the human organism with regard to their amounts. When needed these precursors can be converted in the kidneys to form the biologically active 1α,25-dihydroxyvitamin D, the so-called D hormone. The biologically active vitamin D regulates among others calcium uptake from the intestine, bone mineralization, and it influences a large number of other metabolic pathways such as, e.g., the insulin system.
Measuring the vitamin D level itself is of little benefit when determining the vitamin D status of a patient because concentrations of vitamin D (vitamin D2 and vitamin D3) fluctuate greatly depending on food uptake. In addition, vitamin D has a relatively short biological half-life in the circulation (24 hours), and it is therefore also for this reason not a suitable parameter for determining the vitamin D status of a patient. The same also applies to physiologically active forms of vitamin D (1,25-dihydroxyvitamin D). These biologically active forms also occur in relatively small and highly fluctuating concentrations compared to 25-hydroxyvitamin D. For all these reasons, the quantification of 25-hydroxyvitamin D in particular is a suitable means to globally analyze the total vitamin D status of a patient.
The binding of 25-hydroxyvitamin D or other vitamin D compounds to the vitamin D-binding protein enormously complicates the determination of vitamin D compounds. All known methods require that the vitamin D compound to be analyzed is released or detached from the complex that it forms with the binding protein. In the following this is referred to as the release of a vitamin D compound from vitamin D-binding protein for the sake of simplification, although of course it can only be released from a complex of vitamin D compound and vitamin D-binding protein and not from the vitamin D-binding protein alone.
Since the vitamin D-binding protein has a high tendency to correctly refold, it is often necessary to firstly release vitamin D compounds and then to separate the vitamin D-binding protein from the vitamin D compounds to be analyzed.
Due to the high clinical importance of 25-hydroxyvitamin D, a large number of methods are known from the literature which allow 25-hydroxyvitamin D to be more or less reliably determined.
Haddad, J. G. et al., J. Clin. Endocrinol. Metab. 33 (1971) 992-995, and Eisman, J. A. et al., Anal. Biochem. 80 (1977) 298-305, for example, describe the determination of 25-hydroxyvitamin D concentrations in blood samples using high performance liquid chromatography (HPLC).
Other approaches for the determination of 25-hydroxyvitamin D are based, among others, on the use of vitamin D-binding proteins like those that are present in milk. Thus Holick, M. F. and Ray, R. (U.S. Pat. No. 5,981,779) and DeLuca et al. (EP 0 583 945) describe vitamin D assays for hydroxyvitamin D and dihydroxyvitamin D which are based on the binding of these substances to vitamin D-binding protein where the concentrations of these substances are determined by means of a competitive test procedure. However, a prerequisite of this method is that vitamin D metabolites to be determined firstly have to be isolated from the original blood or serum samples and have to be purified by, for example, chromatography.
Armbruster, F. P. et al. (WO 99/67211) teach that a serum or plasma sample should be prepared for vitamin D determination by ethanol precipitation. In this method the protein precipitate is removed by centrifugation, and the ethanolic supernatant contains soluble vitamin D metabolites. These can be measured in a competitive binding assay.
Alternatively, EP 753,743 teaches that the proteins can be separated from blood or serum samples using a periodate salt. In this case, vitamin D compounds are determined in the protein-free supernatant from the samples treated with periodate. In some commercial tests, acetonitrile is recommended for the extraction of serum or plasma sample (e.g., in the radioimmunoassay from DiaSorin or in the vitamin D test from the Immundiagnostik company).
In recent years a number of different release reagents were proposed which should in principle be suitable for releasing vitamin D compounds from binding protein present in the sample. However, this release or detachment should be carried out under relatively mild conditions, thus enabling a direct use of the sample treated with the release reagent in a binding test (see, for example, WO 02/57797 and US 2004/0132104). Despite immense efforts in recent years, all available methods for determining vitamin D have disadvantages such as laborious sample preparation, poor standardization, poor agreement between test procedures, or bad recovery of spiked vitamin D (see for this in particular Zerwekh, J. E., supra).
It is particularly difficult to automate a test for a vitamin D compound. The automation requires solving a very difficult problem, i.e., surviving a tightrope walk. On the one hand it is necessary to release the vitamin D compounds from vitamin D-binding protein with the aid of a suitable release reagent; on the other hand, the conditions have to be selected such that the sample can be directly analyzed further. A prerequisite of this direct further analysis is that, on the one hand, the vitamin D-binding protein does not bind or no longer to a significant extent binds to the vitamin D compounds during this analysis and thus does not interfere with this analysis and, on the other hand, that the release reagent used does not interfere with the binding of detection reagents such as antibodies to the vitamin D-binding protein to be examined. In addition, it is known that different alleles of the vitamin D-binding protein are present in the human population which behave biochemically differently. The release and measurement of vitamin D compounds should be comparable for various alleles/phenotypes.
Thus the object of the present invention was to develop a release reagent for vitamin D compounds and in particular for hydroxyvitamin D compounds which can at least partially overcome the problems of the prior art. A suitable reagent composition for releasing vitamin D compounds, a method for determining 25-hydroxyvitamin D compounds, the use of the reagent composition, and kits for the determination of 25-hydroxyvitamin D compounds using this reagent composition are described in the following and are encompassed by the attached claims.